During cell division in animal cells the actomyosin cytoskeleton undergoes a dramatic reorganization to form the contractile ring. The formation of this "organelle" is tightly controlled both in a temporal (at the end of mitosis) and spatial (on the metaphase plane) manner. To date there is no clear understanding of the molecular mechanisms that control cytokinesis. The major goal of this proposal is to understand the molecular basis of cytoskeletal reorganization during cytokinesis. We will use Dictyostelium discoideum as a model system because this organism offers many advantages for the studies discussed here. In addition to undergoing cytokinesis very much like animal cells (unlike yeast cells), Dictyostelium is very malleable both at the biochemical and genetic level and its cytoskeleton has been very well characterized. This proposal is aimed at answering the following questions: 1. How is myosin localized in the contractile ring? The mechanisms of myosin localization in the contractile ring will be elucidated. The portion of the myosin molecule required for localization in the contractile ring will be determined. The phosphorylation state of myosin heavy and light chains during the cell cycle will be analyzed. 2. What constitutes a contractile ring? The molecular components of the contractile ring will be identified and their functions analyzed. Contractile rings will be purified and their protein components characterized. The contractile-ring proteins will be cloned and sequenced. The function of these proteins will be assessed by gene- knockout and overexpression experiments. 3. What other genes are involved in cytokinesis? A genetic approach to cytokinesis will be initiated. Dictyostelium mutants defective in cytokinesis will be isolated. The cytokinesis mutants will be characterized. The organization of the actomyosin cytoskeleton will be determined in the isolated mutants. The affected genes will be cloned by DNA-mediated complementation.